Role of sialidase Neu3 and ganglioside GM3 in cardiac fibroblasts activation.

Cardiac fibrosis is a key physiological response to cardiac tissue injury to protect the heart from wall rupture. However, its progression increases heart stiffness, eventually causing a decrease in heart contractility. Unfortunately, to date, no efficient antifibrotic therapies are available to the clinic. This is primarily due to the complexity of the process, which involves several cell types and signaling pathways. For instance, the transforming growth factor beta (TGF-ß) signaling pathway has been recognized to be vital for myofibroblasts activation and fibrosis progression. In this context, complex sphingolipids, such as ganglioside GM3, have been shown to be directly involved in TGF-ß receptor 1 (TGF-R1) activation. In this work, we report that an induced up-regulation of sialidase Neu3, a glycohydrolytic enzyme involved in ganglioside cell homeostasis, can significantly reduce cardiac fibrosis in primary cultures of human cardiac fibroblasts by inhibiting the TGF-ß signaling pathway, ultimately decreasing collagen I deposition. These results support the notion that modulating ganglioside GM3 cell content could represent a novel therapeutic approach for cardiac fibrosis, warranting for further investigations.

Drivers of recombinant soluble influenza A virus hemagglutinin and neuraminidase expression in mammalian cells.

Recombinant soluble trimeric influenza A virus hemagglutinins (HA) and tetrameric neuraminidases (NAs) have proven to be excellent tools to decipher biological properties. Receptor binding and sialic acid cleavage by recombinant proteins correlate satisfactorily compared to whole viruses. Expression of HA and NA can be achieved in a plethora of different laboratory hosts. For immunological and receptor interaction studies however, insect and mammalian cell expressed proteins are preferred due to the presence of N-linked glycosylation and disulfide bond formation. Because mammalian-cell expression is widely applied, an increased expression yield is an important goal. Here we report that using codon-optimized genes and sfGFP fusions, the expression yield of HA can be significantly improved. sfGFP also significantly increased expression yields when fused to the N-terminus of NA. In this study, a suite of different hemagglutinin and neuraminidase constructs are described, which can be valuable tools to study a wide array of different HAs, NAs and their mutants.

Role of Neuraminidase-Producing Bacteria in Exposing Cryptic Carbohydrate Receptors for Streptococcus gordonii Adherence.

Streptococcus gordonii is an early colonizer of the oral cavity. Although a variety of S. gordonii adherence mechanisms have been described, current dogma is that the major receptor for S. gordonii is sialic acid. However, as many bacterial species in the oral cavity produce neuraminidase that can cleave terminal sialic acid, it is unclear whether S. gordonii relies on sialic acid for adherence to oral surfaces or if this species has developed alternative binding strategies. Previous studies have examined adherence to immobilized glycoconjugates and identified binding to additional glycans, but no prior studies have defined the contribution of these different glycan structures in adherence to oral epithelial cells. We determined that the majority of S. gordonii strains tested did not rely on sialic acid for efficient adherence. In fact, adherence of some strains was significantly increased following neuraminidase treatment. Further investigation of representative strains that do not rely on sialic acid for adherence revealed binding not only to sialic acid via the serine-rich repeat protein GspB but also to ß-1,4-linked galactose. Adherence to this carbohydrate occurs via an unknown adhesin distinct from those utilized by Streptococcus oralis and Streptococcus pneumoniae Demonstrating the potential biological relevance of binding to this cryptic receptor, we established that S. oralis increases S. gordonii adherence in a neuraminidase-dependent manner. These data suggest that S. gordonii has evolved to simultaneously utilize both terminal and cryptic receptors in response to the production of neuraminidase by other species in the oral environment.

A synbio approach for selection of highly expressed gene variants in Gram-positive bacteria.

BACKGROUND: The market for recombinant proteins is on the rise, and Gram-positive strains are widely exploited for this purpose. Bacillus subtilis is a profitable host for protein production thanks to its ability to secrete large amounts of proteins, and Lactococcus lactis is an attractive production organism with a long history in food fermentation. RESULTS: We have developed a synbio approach for increasing gene expression in two Gram-positive bacteria. First of all, the gene of interest was coupled to an antibiotic resistance gene to create a growth-based selection system. We then randomised the translation initiation region (TIR) preceding the gene of interest and selected clones that produced high protein titres, as judged by their ability to survive on high concentrations of antibiotic. Using this approach, we were able to significantly increase production of two industrially relevant proteins; sialidase in B. subtilis and tyrosine ammonia lyase in L. lactis. CONCLUSION: Gram-positive bacteria are widely used to produce industrial enzymes. High titres are necessary to make the production economically feasible. The synbio approach presented here is a simple and inexpensive way to increase protein titres, which can be carried out in any laboratory within a few days. It could also be implemented as a tool for applications beyond TIR libraries, such as screening of synthetic, homologous or domain-shuffled genes.

Antitumor effect of the Newcastle disease viral hemagglutinin-neuraminidase gene is expressed through an oncolytic adenovirus effect in osteosarcoma cells.

Newcastle disease virus (NDV) can specifically kill cancer cells and has less toxicity to normal cells. The hemagglutinin-neuraminidase (HN) protein is an important structural protein in NDV pathogenesis and has been postulated as a promising candidate for antitumor therapy. The aim of this study was to investigate the anticancer potential of recombinant adenovirus Ad-HN-PEG3p-E1a. An MTS assay was performed to determine viral proliferation after viral infection, the data showed that the proliferation ability of osteosarcoma cells decreased, whereas there was no significant change in normal hepatic cells. DAPI and Annexin V experiments showed that osteosarcoma cells were killed because of apoptosis, active oxygen content, and augmented mitochondrial membrane potential loss. Caspase Activity Assay Kits were used to detect the caspase-3 activities of the treated OS-732 for increased expression. Western blot analysis showed that cytochrome C increased significantly and apoptosis of the virus was confirmed in tumor cells. In-vivo experiments show that NDV has an inhibitory effect on tumor growth. The recombinant adenovirus, which is composed of a HN protein and progressive increment promoter PEG3p, could inhibit the growth of OS-732 and promote the apoptosis of tumor cells. However, there was no clear relationship with normal cell (L02) apoptosis.

NanR Regulates nanI Sialidase Expression by Clostridium perfringens F4969, a Human Enteropathogenic Strain.

Clostridium perfringens can produce up to three different sialidases, including NanI, its major exosialidase. The current study first showed that human intestinal strains of C. perfringens can grow by utilizing either glucose or sialic acids, such as N-acetylneuraminic acid (Neu5Ac), which are the end products of sialidase activity. For the human enteropathogenic strain F4969, it was then determined that culture supernatant sialidase activity and expression of exosialidase genes, particularly nanI, are influenced by the presence of Neu5Ac or glucose. Low Neu5Ac concentrations increased culture supernatant sialidase activity, largely by stimulating nanI transcription. In contrast, low glucose concentrations did not affect exosialidase activity or nanI transcription. However, either high Neu5Ac or high glucose concentrations repressed F4969 culture supernatant sialidase activity and nanI transcription levels. Furthermore, high glucose levels repressed F4969 culture sialidase activity and nanI expression even in the presence of low Neu5AC concentrations. To begin to evaluate the mechanistic basis for nanI expression, a nanR null mutant was used to demonstrate that NanR, a member of the RpiR family of regulatory proteins, decreases exosialidase activity and nanI transcription in the absence of sialic acid. The ability of C. perfringens to regulate its exosialidase activity, largely by controlling nanI expression, may affect intestinal pathogenesis by affecting the production of NanI, which may affect C. perfringens growth, adhesion, and toxin binding in vivo.

Manipulation of neuraminidase packaging signals and hemagglutinin residues improves the growth of A/Anhui/1/2013 (H7N9) influenza vaccine virus yield in eggs.

In 2013, a novel avian-origin H7N9 influenza A virus causing severe lower respiratory tract disease in humans emerged in China, with continued sporadic cases. An effective vaccine is needed for this virus in case it acquires transmissibility among humans; however, PR8-based A/Anhui/1/2013 (Anhui/1, H7N9), a WHO-recommended H7N9 candidate vaccine virus (CVV) for vaccine production, does not replicate well in chicken eggs, posing an obstacle to egg-based vaccine production. To address this issue, we explored the possibility that PR8's hemagglutinin (HA) and neuraminidase (NA) packaging signals mediate improvement of Anhui/1 CVV yield in eggs. We constructed chimeric HA and NA genes having the coding region of Anhui/1 HA and NA flanked by the 5' and 3' packaging signals of PR8's HA and NA, respectively. The growth of CVVs containing the chimeric HA was not affected, but that of those containing the chimeric NA gene grew in embryonated chicken eggs with a more than 2-fold higher titer than that of WT CVV. Upon 6 passages in eggs further yield increase was achieved although this was not associated with any changes in the chimeric NA gene. The HA of the passaged CVV, did, however, exhibit egg-adaptive mutations and one of them (HA-G218E) improved CVV growth in eggs without significantly changing antigenicity. The HA-G218E substitution and a chimeric NA, thus, combine to provide an Anhui/1 CVV with properties more favorable for vaccine manufacture.

Cloning, expression, and characterization of a novel sialidase from Brevibacterium casei.

The sialidase gene from Brevibacterium casei was cloned in pET28a and overexpressed as a histidine-tagged protein in Escherichia coli BL21(DE3). The histidine-tagged sialidase protein was purified and characterized from the crude cell extracts of isopropyl-ß-d-thiogalactopyranoside-induced cells using Ni-NTA agarose chromatography. SDS-PAGE using the purified sialidase indicated a single band at 116 kDa. This sialidase showed maximum activity at a pH of 5.5 and temperature of 37 °C. The kinetic parameters Km and Vmax for the artificial substrate 2'-(4-methylumbelliferyl)-α-d-N-acetyl-neuraminic acid sodium salt hydrate were 1.69 × 10-3 mM and 244 mmol·Min-1 ·mg-1 , respectively. The sialidase may catalyze the hydrolysis of terminal sialic acids linked by the α-(2,3) and α-(2,8) linkage of polysialogangliosides, but it does not act on monosialotetrahexosylganglioside (GM1), which offers it a great potential for commercially producing GM1 from polysialogangliosides.

Sialylation and desialylation dynamics of monocytes upon differentiation and polarization to macrophages.

Sialic acids (SAs) often exist as the terminal sugars of glycan structures of cell surface glycoproteins and glycolipids. The level and linkages of cell surface SAs, which are controlled by both sialylation and desialylation processes and environment cues, can dramatically impact cell properties and represent different cellular status. In this study, we systematically examined the sialylation and desialylation profiles of THP-1 monocytes after differentiation to M0 macrophages, and polarization to M1 and M2 macrophages by the combination of LC-MS/MS, flow cytometry and confocal microscopy. Interestingly, both α2-3- and α2-6-linked SAs on the cell surface decreased after monocytes were differentiated to macrophages, which was in accordance with the increased level of free SA in the cell culture medium and the elevated activity of endogenous Neu1 sialidase. Meanwhile, the siaoglycoconjugates inside the cells increased as confirmed by confocal microscopy and the total SA inside the cells increased as determined by LC-MS/MS. Western blot analysis showed higher expression levels of sialyltransferases, including ST3Gal-I, ST3Gal-V, ST6Gal-I and ST6GalNAc-II. Further, upon polarization, the cell surface sialylation levels of M1 and M2 macrophages remained the same as M0 macrophages, while a slight decrease of cellular SAs in the M1 macrophages but increase in the M2 macrophages were confirmed by LC-MS/MS.

Glycosomal bromodomain factor 1 from Trypanosoma cruzi enhances trypomastigote cell infection and intracellular amastigote growth.

Acetylation is a ubiquitous protein modification present in prokaryotic and eukaryotic cells that participates in the regulation of many cellular processes. The bromodomain is the only domain known to bind acetylated lysine residues. In the last few years, many bromodomain inhibitors have been developed in order to treat diseases caused by aberrant acetylation of lysine residues and have been tested as anti-parasitic drugs. In the present paper, we report the first characterization of Trypanosoma cruzi bromodomain factor 1 (TcBDF1). TcBDF1 is expressed in all life cycle stages, but it is developmentally regulated. It localizes in the glycosomes directed by a PTS2 (peroxisome-targeting signal 2) sequence. The overexpression of wild-type TcBDF1 is detrimental for epimastigotes, but it enhances the infectivity rate of trypomastigotes and the replication of amastigotes. On the other hand, the overexpression of a mutated version of TcBDF1 has no effect on epimastigotes, but it does negatively affect trypomastigotes' infection and amastigotes' replication.

Upregulation of sialidase NEU3 in head and neck squamous cell carcinoma associated with lymph node metastasis.

Regional lymph node metastasis in head and neck squamous cell carcinoma (HNSCC) is a crucial event for its progression, associated with a high rate of mortality. Sialidase, a key enzyme for the regulation of cellular sialic acids through catalyzing the initial step of degradation of glycoproteins and glycolipids, has been implicated in cancer progression. To facilitate the development of novel treatments for HNSCC, we have investigated whether sialidase is involved in the progression of this cancer. We found plasma membrane-associated sialidase (NEU3) to be significantly upregulated in tumor compared to non-tumor tissues; particularly, an increase in its mRNA levels was significantly associated with lymph node metastasis. To understand the mechanisms, we analyzed the NEU3-mediated effects on the malignant phenotype using squamous carcinoma HSC-2 and SAS cells. NEU3 promoted cell motility and invasion, accompanied by the increased expression of MMP-9, whereas NEU3 silencing or the activity-null mutant did not. NEU3 enhanced phosphorylation of epidermal growth factor receptor (EGFR), and an EGFR inhibitor, AG1478, abrogated the NEU3-induced MMP9 augmentation. These findings identify NEU3 as a participant in HNSCC progression through the regulation of EGFR signaling and thus as a potential target for inhibiting EGFR-mediated tumor progression.

Structural Analysis of the Roles of Influenza A Virus Membrane-Associated Proteins in Assembly and Morphology.

UNLABELLED: The assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue culture-adapted strains but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), and M2 ion channel all contribute to virus assembly. When expressed individually or in combination in cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release, and morphology remain unclear. To investigate these roles, we produced membrane-enveloped particles by plasmid-derived expression of combinations of HA, NA, and M proteins (M1 and M2) or by infection with influenza A virus. We monitored particle release, particle morphology, and plasma membrane morphology by using biochemical methods, electron microscopy, electron tomography, and cryo-electron tomography. Our data suggest that HA, NA, or HANA (HA plus NA) expression leads to particle release through nonspecific induction of membrane curvature. In contrast, coexpression with the M proteins clusters the glycoproteins into filamentous membrane protrusions, which can be released as particles by formation of a constricted neck at the base. HA and NA are preferentially distributed to differently curved membranes within these particles. Both the budding intermediates and the released particles are morphologically similar to those produced during infection with influenza A virus. Together, our data provide new insights into influenza virus assembly and show that the M segment together with either of the glycoproteins is the minimal requirement to assemble and release membrane-enveloped particles that are truly virus-like. IMPORTANCE: Influenza A virus is a major respiratory pathogen. It assembles membrane-enveloped virus particles whose shapes vary from spherical to filamentous. Here we examine the roles of individual viral proteins in mediating virus assembly and determining virus shape. To do this, we used a range of electron microscopy techniques to obtain and compare two- and three-dimensional images of virus particles and virus-like particles during and after assembly. The virus-like particles were produced using different combinations of viral proteins. Among our results, we found that coexpression of one or both of the viral surface proteins (hemagglutinin and neuraminidase) with the viral membrane-associated proteins encoded by the M segment results in assembly and release of filamentous virus-like particles in a manner very similar to that of the budding and release of influenza virions. These data provide novel insights into the roles played by individual viral proteins in influenza A virus assembly.

High Production of Neuraminidase by a Vibrio cholerae Non-O1 Strain--the First Possible Alternative to Toxigenic Producers.

Vibrio cholerae neuraminidase (VCNA) is widely used in biochemical and medical research, in processes for preparing homogenous sialoconjugates, and in the pharmaceutical industry. Its production by non-toxigenic strains is quite desirable, in order to avoid the expensive safety measures. Here, we report the first method for highly effective production of a novel, purified V. cholerae extracellular neuraminidase from a non-toxigenic strain. The enzyme is highly active, and its properties, as well as the responsible gene nanH, are practically identical with those of the toxigenic strains. It cleaves α,2 → 3 and α,2 → 6 glycosidic bonds with highest affinity (K M 1.7 × 10(-5) µM) for human transferrin. The deduced amino acid sequence of the enzyme reveals three binding sites for Ca(2+) and one for sialic acid. The sequence analysis of the nanH gene, being the first for a V. cholerae non-O1 strain, shows 99% identity with a new nanH allele of an O1 Vibrio strain. The simple laboratory technology for efficient production of the new VCNA is based on the use of common and cheap nutrient media and easily available inducer--glycomacropeptide. The rapid purification consists of salting-out and diethylaminoethanol (DEAE) and Q-Sepharose chromatography steps. Purified preparation contains no aldolase and protease, which gives the production scheme a great potential for industrial application.

Increased sialidase activity in serum of cancer patients: Identification of sialidase and inhibitor activities in human serum.

Aberrant sialylation in glycoproteins and glycolipids is a characteristic feature of malignancy. Human sialidases, which catalyze the removal of sialic acid residues from glycoconjugates, have been implicated in cancer progression. They have been detected in a wide variety of human cells and tissues, but few studies have focused on their existence in human serum. Among the four types identified to date, we previously demonstrated that plasma membrane-associated ganglioside sialidase (NEU3) is markedly upregulated in various human cancers, including examples in the colon and prostate. Here, using a sensitive assay method, we found a significant increase of sialidase activity in the serum of patients with prostate cancer compared with that in healthy subjects having low activity, if any. Activity was apparent with gangliosides as substrates, but only to a very limited extent with 4-methylumbelliferyl sialic acid, a good synthetic substrate for sialidases other than human NEU3. The serum sialidase was also almost entirely immunoprecipitated with anti-NEU3 antibody, but not with antibodies for other sialidases. Interestingly, sera additionally contained inhibitory activity against the sialidase and also against recombinant human NEU3. The sialidase and inhibitor activities could be separated by exosome isolation and by hydrophobic column chromatography. The serum sialidase was assessed by a sandwich ELISA method using two anti-NEU3 antibodies. The results provide strong evidence that the serum sialidase is, in fact, NEU3, and this subtype may, therefore, be a potential utility for novel diagnosis of human cancers.

A miRNA signature of chemoresistant mesenchymal phenotype identifies novel molecular targets associated with advanced pancreatic cancer.

In this study a microRNA (miRNA) signature was identified in a gemcitabine resistant pancreatic ductal adenocarcinoma (PDAC) cell line model (BxPC3-GZR) and this signature was further examined in advanced PDAC tumor specimens from The Cancer Genome Atlas (TCGA) database. BxPC3-GZR showed a mesenchymal phenotype, expressed high levels of CD44 and showed a highly significant deregulation of 17 miRNAs. Based on relevance to cancer, a seven-miRNA signature (miR-100, miR-125b, miR-155, miR-21, miR-205, miR-27b and miR-455-3p) was selected for further studies. A strong correlation was observed for six of the seven miRNAs in 43 advanced tumor specimens compared to normal pancreas tissue. To assess the functional relevance we initially focused on miRNA-125b, which is over-expressed in both the BxPC3-GZR model and advanced PDAC tumor specimens. Knockdown of miRNA-125b in BxPC3-GZR and Panc-1 cells caused a partial reversal of the mesenchymal phenotype and enhanced response to gemcitabine. Moreover, RNA-seq data from each of 40 advanced PDAC tumor specimens from the TCGA data base indicate a negative correlation between expression of miRNA-125b and five of six potential target genes (BAP1, BBC3, NEU1, BCL2, STARD13). Thus far, two of these target genes, BBC3 and NEU1, that are tumor suppressor genes but not yet studied in PDAC, appear to be functional targets of miR-125b since knockdown of miR125b caused their up regulation. These miRNAs and their molecular targets may serve as targets to enhance sensitivity to chemotherapy and reduce metastatic spread.

Construction of recombinant Marek's disease virus (MDV) lacking the meq oncogene and co-expressing AIV-H9N2 HA and NA genes under control of exogenous promoters.

To develop a recombinant Marek's disease virus (rMDV1) co-expressing the hemagglutinin gene (HA) and neuramidinase gene (NA) from a low pathogenic avian influenza virus (LPAIV) H9N2 strain and lacking the meq oncogene that shares homology with the Jun/Fos family of transcriptional factors, a wild strain of MDV GX0101 was used as parental virus, the HA and NA genes co-expression cassette under control of the CMV and SV40 early promoters was inserted at two meq sites of GX0101 to form a new meq knock-out mutant MDV (MZC12HA/NA) through homologous recombination. MZC12HA/NA was reconstituted by transfection of recombinant BAC-MDV DNA into the secondary chicken embryo fibroblast (CEF) cells. Highly purified MZC12HA/NA was obtained after four rounds of plaque purification and proliferation. In vitro growth properties of recombinant virus were also inspected and concluded that the MZC12HA/NA had the same growth kinetics in CEF cultures as its parental wild type virus GX0101. Southern blot indicated that co-expression cassette was successfully inserted at two copies sites of meq gene, so two meq genes were knocked-out completely. RT-qPCR showed transcription and expression levels of the HA and NA genes were both significantly higher than that of GX0101 own pp38 gene. Indirect fluorescence antibody (IFA) test, and Western blot analyses indicated that HA and NA genes were co-expressed simultaneously under control of the different promoters but meq genes were not. These results herald a new and effective recombinant meq-deleted MDV-based AIV-H9N2 vaccine may be useful in protecting chickens from very virulent MDV and H9N2 challenges.

Expression of functional recombinant hemagglutinin and neuraminidase proteins from the novel H7N9 influenza virus using the baculovirus expression system.

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Regulation of sialidase production in Clostridium perfringens by the orphan sensor histidine kinase ReeS.

Clostridium perfringens is ubiquitous in nature and is often found as a commensal of the human and animal gastrointestinal tract. It is the primary etiological agent of clostridial myonecrosis, or gas gangrene, a serious infection that results in extensive tissue necrosis due to the action of one or more potent extracellular toxins. α-toxin and perfringolysin O are the major extracellular toxins involved in the pathogenesis of gas gangrene, but histotoxic strains of C. perfringens, such as strain 13, also produce many degradative enzymes such as collagenases, hyaluronidases, sialidases and the cysteine protease, α-clostripain. The production of many of these toxins is regulated either directly or indirectly by the global VirSR two-component signal transduction system. By isolating a chromosomal mutant and carrying out microarray analysis we have identified an orphan sensor histidine kinase, which we have named ReeS (regulator of extracellular enzymes sensor). Expression of the sialidase genes nanI and nanJ was down-regulated in a reeS mutant. Since complementation with the wild-type reeS gene restored nanI and nanJ expression to wild-type levels, as shown by quantitative reverse transcription-PCR and sialidase assays we concluded that ReeS positively regulates the expression of these sialidase genes. However, mutation of the reeS gene had no significant effect on virulence in the mouse myonecrosis model. Sialidase production in C. perfringens has been previously shown to be regulated by both the VirSR system and RevR. In this report, we have analyzed a previously unknown sensor histidine kinase, ReeS, and have shown that it also is involved in controlling the expression of sialidase genes, adding further complexity to the regulatory network that controls sialidase production in C. perfringens.

[Expression of NA of influenza virus and C3d fusion gene in replication-defective recombinant adenovirus and its immune efficacy analysis].

OBJECTIVE: To construct a replication-defective recombinant adenovirus expressing the fusion gene of neuraminidase (NA) gene in influenza virus A/FM/1/47 and C3d and to evaluate the induced immune efficacy. METHODS: NA-C3d was cloned into shutter vector pAdTrack-CMV, which was cotransformated with adenovirus DNA into E. coli BJ5183. The recombinant adenovirus genomic DNA was generated through homological recombination. The recombinant adenovirus was produced by transfecting 293 cell line with the genomic DNA and the induced immune efficacy in mice were analyzed. RESULTS: The integration of NA-C3d in the adenovirus genomic DNA and its expression were confirmed by PCR and Western-Blot assays respectively. After intranasal immunization, the serum IgG was induced at a titer of 1: 1000 and 1:100 000 in BALB/c mice at primary and secondary immunization respectively. The vaccinated mice were completely survived when challenged with wide influenza virus. CONCLUSION: recombinant adenovirus expressing NA-C3d was successfully constructed and it could induce desired immune efficacy.

Regulation of neuraminidase expression in Streptococcus pneumoniae.

BACKGROUND: Sialic acid (N-acetylneuraminic acid; NeuNAc) is one of the most important carbohydrates for Streptococcus pneumoniae due of its role as a carbon and energy source, receptor for adhesion and invasion and molecular signal for promotion of biofilm formation, nasopharyngeal carriage and invasion of the lung. RESULTS: In this work, NeuNAc and its metabolic derivative N-acetyl mannosamine (ManNAc) were used to analyze regulatory mechanisms of the neuraminidase locus expression. Genomic and metabolic comparison to Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii and Streptococcus sanguinis elucidates the metabolic association of the two amino sugars to different parts of the locus coding for the two main pneumococcal neuraminidases and confirms the substrate specificity of the respective ABC transporters. Quantitative gene expression analysis shows repression of the locus by glucose and induction of all predicted transcriptional units by ManNAc and NeuNAc, each inducing with higher efficiency the operon encoding for the transporter with higher specificity for the respective amino sugar. Cytofluorimetric analysis demonstrated enhanced surface exposure of NanA on pneumococci grown in NeuNAc and ManNAc and an activity assay allowed to quantify approximately twelve times as much neuraminidase activity on induced cells as opposed to glucose grown cells. CONCLUSIONS: The present data increase the understanding of metabolic regulation of the nanAB locus and indicate that experiments aimed at the elucidation of the relevance of neuraminidases in pneumococcal virulence should possibly not be carried out on bacteria grown in glucose containing media.